抗中华眼镜蛇毒鸡卵黄抗体的制备及其效价测定

目的探索免疫鸡制备高效价抗眼镜蛇毒抗体的新方法。方法用中华眼镜蛇原毒作抗原免疫22周龄的莱航母鸡,水溶法粗提抗体,DEAE Sepharos FF柱纯化,切向流超滤膜脱盐及浓缩,免疫电泳及双向免疫扩散法进行鉴定及效价测定,采用BCATMProte in Assay K it测定蛋白含量。结果鸡卵黄经水溶法的粗提物与中华眼镜蛇毒即有较明显沉淀反应,其效价随着纯度的提高而增强。将马源性抗血清的蛋白质含量调至与浓缩的IgY相同(2mg/m l),经双向免疫扩散及免疫电泳鉴定,该抗体不但对中华眼镜蛇毒有特异性结合,与孟加拉眼镜蛇毒亦有较强的交叉免疫活性,其效价较马抗眼镜蛇毒血清高4倍以上。结论用中华眼镜蛇原毒制备的IgY抗体,其效价较马抗血清有显著提高,并与孟加拉眼镜蛇毒有高度交叉免疫。本实验为抗眼镜蛇IgY的应用及其它抗蛇毒IgY的制备奠定了基础。

Preparation and titration for venom of Naja atra antibodies from chicken egg yolk

Objective Explore the new method of generating high titer antibody of anti-cobra venom through immunizing hens. Methods Use the original venom of Naja atra to immunize 22 week's Leghorn hens.Immunoglobulin yolk(IgY) was separated from egg yolk by water dilution method and purified by anion chromatography of DEAE Sepharos FF,then desalinated and concentrated by flip flow filtration.Immunoelectrophoresis technique and double immunodiffusion was used to identify and measure the titer of IgY,and protein concentration was measured by BCA~(TM) Protein Assay Kit method. Results The IgY solution obtained from water dilution method has specific binding with Naja atra and the titration increased coinciding with purity.When adjusted the protein concentration to the same as anti-cobra venom from horse serum(2mg/ml),the IgY not only had specific binding with Naja atra,but also had highly cross immunocompetence with Naja kaouthia.The titration was higher for more than 4 folds than anti-cobra venom rom horse serum. Conclusion IgY produced by the original venom of Naja atra has obviously higher titer than anti-cobra venom from horse serum,and has highly cross immunocompetence with Naja kaouthia.This research lays a foundation for application of IgY and preparation for other anti-snake venom.